Evaluation of MicroRNA-99a and MicroRNA-205 Expression Levels in Bladder Cancer

Bladder cancer is the second most common cancer in the genitourinary tract, showing often recurrence and progress into invasive states. Epigenetic changes, such as microRNA alteration are involved in bladder cancer tumorigenesis through a variety of signaling pathways. The epigenetic state depends on geographic and lifestyle conditions. The aim of this study was to investigate the expression level of microRNA-99a and microRNA-205 in bladder cancer in Iranian populations and to determine the relationship between their expressions with clinicophatological features. 36 patients with bladder cancer were included in the study. The control group was the healthy adjacent tissue of the same patients. Total RNA was extracted from approximately 50 mg tissue using TRIzol reagent. cDNA was synthesized and Real-Time PCR was carried out using specific primers. The Unisp6 rRNA was used as a reference gene. A significant decrease was found in the expression level of miR-99a in tumor samples, compared to healthy adjacent tissues (P<0.001). The increased expression level of miR-99a was significantly associated with muscle invasion (P=0.02). The receiver operating characteristic (ROC) analysis for miR-99a showed AUC value equal to 0.944, with specificity of 97%, sensitivity of 91%, and cut off value of 8.31 (P<0.001). A significant association was found between smoking and miR-99a (P=0.04) and miR-205 (P= 0.01) expression levels. Dramatic down-regulation of miR-99a in bladder cancer tissues confirmed the tumor suppressor role of miR-99a in bladder cancer. A higher amount of miR-99a expression was associated with invasive bladder cancer. According to ROC analysis, miR-99a could be considered as a valuable diagnostic biomarker.

respectively (2). It was estimated that 2.4% of the men and women face BC diagnosis throughout their lives (3). In spite of the high incidence and prevalence, treatment of bladder cancer has not changed in the nearly recent three decades (4).
Cystoscopy and transurethral resection, considered as invasive processes, are generally applied for diagnosis, treatment and follow-up of patients (5).
By 2025, up to 45% increase of cancer incidence is expected in developing countries (6). Currently, there is no standard screening test to identify individuals at risk of BC (2). Carcinogenesis and progression of BC are caused by multiple genetic and epigenetic changes (7). Given the foregoing, the accurate mechanism of bladder carcinogenesis has not clearly been determined to date. Therefore, there is a need for understanding the process of genetic and epigenetic alterations to identify molecular biomarkers for diagnosis, prognosis and therapeutic decision (8).
MicroRNAs (MiRNAs) are a group of short (approximately 18-22 nucleotides) non-coding single-stranded RNA molecules that normally serve as a negative regulator of gene expression during different biologic processes including cell differentiation, proliferation, death, metabolism, apoptosis, carcinogenesis, immune response and energy homeostasis (9)(10)(11). Some miRNAs act as tumor suppressors while others serve as oncogenes during cancer development and progression (12)(13)(14). MiRNA expression in BC was determined for the first time, by Gottardo et al. (13). smoking and recurrence are given in Table 1.

Follow-up protocol
All patients were followed up until August, 2016 every 3 months via urine cytology and ultrasonography. Transurethral resection was applied when abnormal cytology or suspected lesion was observed. Out of 36 patients, 4 died during follow up periods, and 16 cases showed recurrence.  Nonetheless, no statistically significant difference was observed between case and control groups regarding miR-205 expression (P=0.38) ( Table 2).

Association between muscle invasion, grading, and miRNA expression
MiR-99a was found to be overexpressed in tumors with MIBC (P= 0.02) (  (Table 3) as well as between miR-205 expression and the grade of the tumor (P= 0.87) ( Table 4).

Association between smoking, carcinogen exposure, recurrence state and miRNA expression
A remarkable down-regulation of miR-99a and miR-205 was found in smoker patients (P= 0.04, P= 0.01, respectively) ( Table 5). In addition, a direct and significant association was detected between smoking and muscle invasion (P= 0.03).
There was a significant association between the expression level of miR-99a in patients exposed to carcinogens such as asbestos and active chemical colors (P=0.04).    (Figure 2).

Discussion
BC is the fourth and third most common cancer in developed countries and arnong Iranian men, respectively(1). The accurate mechanism of bladder tumorigenesis has not been clearly determined    (17,20).
Several studies showed that miR-99a expression often decreases in most cases with prostate, bladder, uterus and lung cancers (18,19,27). Such a reduction was observed in almost all samples (35 out of 36) in our study.
Approximately 75% of bladder tumors were shown to be superficial at diagnosis times, meaning that the muscle is not involved, and the remaining 25 % invade the muscle (28). In the present study, 64 % and 36% of the patients were diagnosed as NMIBC and MIBC, respectively, which was similar to other studies. The miR-99a expression level in MIBC was higher than that in NMIBC, confirming the results of Wszolek's investigation (29).
In comparison with low grade and papillary urothelial neoplasm of low malignant potential, miR-99a was overexpressed in high grade in our study, supporting its role in poor differentiation of tumor cells. In some cases, due to insufficient tissue specimens, misclassification of malignant samples was obtained by biopsy, in which the pathologist was unable to verify the correct conditions (22).
Therefore, overexpression of miR-99a can be useful in assessing tumor invasion.
Considering the tumor suppressor role of miR-99a, its down regulation is expected with cancer progression. According to our results, miR-99a was found to be underexpressed in the early stage of BC, while increased miR-99a expression was detected in muscle invasive and high-grade tumors.